This proposal investigates the regulation and function of two stress- inducible proteins GRP78/BiP and GRP94. As Ca2+ binding molecular chaperones localized in the endoplasmic reticulum (ER), they play important roles in protein folding, secretion and can confer protection against disruption of Ca2+ homeostasis. In tumor cells, induction of the GRPs correlates with resistance to cell mediated cytotoxicity. Since stress in the ER such as Ca2+ depletion or protein malfolding can activate the grp78 and grp94 promoters, the GRP system offers a unique model to study intra-organelle signaling. To dissect biochemically the regulatory pathways leading to grp induction in mammalian cells, our first set of specific aims will focus on elucidating the transcriptional machinery that acts on the stress response elements of the grp78 and grp94 promoter. Specifically, we will continue experiments now in progress to examine the interdependence of several transcription factors that bind to the control sites. Both in vivo and in vitro assays will be performed to test the individual and collective contribution of the interacting factors. Stress induced phosphorylation changes will also be examined. The main hypothesis to be tested is that the grp promoter system utilizes the multimeric CCAAT binding protein CBF to stabilize grp specific transcription factors such as p7OCORE. In specific cell types, the evolutionarily conserved dbpA/B proteins implicated in the E. coli cold stress response may also regulate the grp stress induction. The studies on phosphorylation can lead to future investigations aimed at dissecting the kinase signaling cascade regulating the grp stress response. The second set of specific aims focuses on the protective function of GRP78 and GRP94 towards apoptosis. This is based on our recent observation that suppression of GRP78 induction in a fibrosarcoma cell line eliminates resistance to TNF and CTL mediated lysis. The hypothesis to be tested is that whether the GRPs can control cellular signaling by regulating TNF receptor and Fas expression on the cell surface and whether they can provide resistance intracellularly. To test definitively the essential functions of GRP78, we propose to perform targeted disruption of this mammalian gene through homologous recombination. The long term goal is to create cell lines and mouse model systems with an inactivated grp78 gene for the study of tumor growth, B and T cell development, immunoglobulin production, and resistance to physiological stress. These studies may also lead to identification of novel targets of GRP function.